Compositions and Methods for Cancer Immunotherapy

ABSTRACT

The invention relates to immunotherapeutic compounds and to methods for stimulating an immune response in a subject individual at risk for developing cancer, diagnosed with a cancer, in treatment for cancer, or in post-therapy recovery from cancer or the compounds of the invention can be administered as a prophylactic to a subject individual to prevent or delay the development of cancer.

FIELD OF THE INVENTION

The invention provides compositions and methods for cancerimmunotherapy.

1. BACKGROUND OF THE INVENTION

Cancer immunotherapy involves the use of compositions and methods toelicit and enhance an individual's own immune system against cancerouscells, or infections that predispose to cancer. Cancer vaccines functionby triggering the immune system to mount a response to an antigen (e.g.,typically a protein, peptide, or carbohydrate) that is introduced intothe body in a non-carcinogenic form and triggers the body to conferimmunity or obtain a long-lived “memory” immune response. See, e.g.,Kast, Peptide-Based Cancer Vaccines, Landes Bioscience (2000); Stern etal, Cancer Vaccines and Immunotherapy, Cambridge University Press(2000). Once the immune system response is established, exposure of theimmune system to this antigen (e.g., in the form of a cancerous tumor)results in a rapid and robust immune response.

It is often necessary to enhance the immune response to the antigenspresent in a vaccine in order to stimulate the immune system to asufficient extent to make a vaccine effective, i.e., to confer immunity.Many protein, peptide and carbohydrate antigens, administered alone, donot generate a sufficient response to confer immunity. The reasons forthis may be that the antigens recognized by cancer reactive immuneresponses originate from proteins that are expressed in normal tissue ofthe same histological type as the cancer, such that immunologictolerance may prevent effective immune responses to the antigens. Suchantigens need to be presented to the immune system in such a way thatthey will generate an immune response. To this end, adjuvants have beendevised which immobilize antigens and enhance the immune response. Thebest known adjuvant, Freund's complete adjuvant, consists of a mixtureof mycobacteria in an oil/water emulsion. Freund's adjuvant works (i) byenhancing cell and humoral-mediated immunity and (ii) by blocking rapiddispersal of the antigen challenge (the depot effect). Freund's adjuvantis used primarily with experimental therapies to help stimulate theimmune system in animals, and in humans the mycobacterial preparationBacille Calmette-Guérin (BCG), is an immunotherapy approved as atreatment for bladder cancer.

Another molecule that has been shown to have immunostimulatory oradjuvant activity is endotoxin, also known as lipopolysaccharide (LPS).LPS is also a model adjuvant that can overcome tolerance to selfantigens. Waldner, et al., J. Clin. Invest., (2004); 113 990-997. WhileLPS is too toxic to be a viable adjuvant, molecules that arestructurally related to endotoxin, such as monophosphoryl lipid A(“MPL”), are being tested as adjuvants in clinical trials. The onlyFDA-approved adjuvant for use in humans are aluminum salts, alum.

There is a need in the art for safe and effective compositions that canstimulate the immune system as a cancer immunotherapeutic. The inventionis directed to this, as well as other, important ends.

2. SUMMARY OF THE INVENTION

The present invention relates to a composition having (i) at least oneimmunotherapeutic agent which may be one or more cancer antigens, one ormore antigens derived from a virus associated with cancer, one or moreanti-cancer antibodies; and an anti-idiotypic antibody to an anti-cancerantibody; and, (ii) one or more compounds of formulae (I), (II), (III),(IV), and (V) and/or a pharmaceutically acceptable salt, hydrate,solvate, stereoisomer, amorphous solid thereof, or any combinationthereof.

The compounds of the invention can administered to a subject individualat risk for developing cancer, diagnosed with a cancer, in treatment forcancer, or in post-therapy recovery from cancer or the compounds of theinvention can be administered as a prophylactic to a subject individualto prevent or delay the development of cancer.

The invention further relates to methods for stimulating an immuneresponse in an subject individual by (a) administering to the individualat least one immunotherapeutic agent selected from one or more cancerantigens; one or more viral derived antigen associated with cancer; oneor more anti-cancer antibodies; and one or more anti-idiotypicantibodies to an anti-cancer antibody; and (b) administering to theindividual one or more compound selected from formulae (I), (II), (III),(IV) and (V) and/or a pharmaceutically acceptable salt, hydrate,solvate, stereoisomer, amorphous solid thereof, or any combinationthereof.

These and other aspects of the invention are described in more detailherein.

3. FIGURES

FIG. 1 is a graph depicting the percentage of tumor-bearing micesurviving after treatment with subcutaneous B16 GM-CSF(R) cells, E6020,or B16 GM-CSF (r) cells and E6020, or with no treatment.

FIG. 2 is a graph depicting the percentage of tumor-bearing micesurviving after intratumoral treatment with B16 GM-CSF (R) cells, or B16GM-CSF(r) cells and E6020, or with no treatment.

FIG. 3 is a graph depicting the percentage of animals without sign ofdisease after no treatment or treatment with vaccine and E6020.

FIG. 4 is a graph depicting the percentage of animals alive after notreatment, treatment with vaccine, or treatment with E6020.

4. DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions comprising (i) at least oneimmunotherapeutic agent selected from one or more cancer antigens, oneor more antigens derived from a virus associated with cancer, one ormore an anti-cancer antibody, and one or more anti-idiotypic antibody toan anti-cancer antibody, and (ii) one or more compounds selected fromformulae (I), (II), (III), (IV), and (V), and/or a pharmaceuticallyacceptable salt, hydrate, solvate, stereoisomer, amorphous solidthereof, or any combination thereof. The compounds are described indetail below. The compositions may be therapeutic, that is, thecompounds are administered to treat an existing cancer, or to preventthe recurrence of a cancer, or prophylactic, that is, the compounds areadministered to prevent or delay the development of cancer. When thecompositions are used therapeutically, they are administered to cancerpatients and are designed to elicit an immune response to stabilize atumor by preventing or slowing the growth of the existing cancer, toprevent the spread of a tumor or of metastases, to reduce the tumorsize, to prevent the recurrence of treated cancer, or to eliminatecancer cells not killed by earlier treatments. A composition used as aprophylactic treatment is administered to individuals who do not havecancer, and are designed to elicit an immune response to targetpotential cancer cells or to target an antigen derived from a virusassociated with cancer.

The compositions of this invention may include more than oneimmunotherapeutic agent with another immunotherapeutic agent, forexample, a cancer antigen in combination with one or more antigensderived from a virus associated with cancer, one or more anti-cancerantibodies, and one or more anti-idiotypic antibodies to an anti-cancerantibody. Another embodiment of the compositions may include one or morecancer antigens and an anti-cancer antibody and/or an anti-idiotypicantibody to an anti-cancer antibody. Other embodiments of thecompositions may include an anti-cancer antibody and an anti-idiotypicantibody to an anti-cancer antibody. Another embodiment may include oneor more antigens derived from a virus associated with cancer and ananti-cancer antibody and/or an anti-idiotypic antibody to an anti-cancerantibody.

4.1 Cancer Antigens

One of the immunotherapeutic agents of the pharmaceutical compositionmay be one or more cancer antigens. A cancer antigen is (a) a cellsurface antigen that can be found on a malignant cell, (b) an antigenthat can be found inside a malignant cell or (c) a mediator of tumorcell growth.

The term “cancer antigen” refers to (i) tumor-specific antigens, (ii)tumor-associated antigens, (iii) cells that express tumor-specificantigens, (iv) cells that express tumor-associated antigens, (v)embryonic antigens on tumors, (vi) autologous tumor cells, (vii)tumor-specific membrane antigens, (viii) tumor-associated membraneantigens, (ix) growth factor receptors, (x) growth factor ligands, and(xi) any other type of antigen or antigen-presenting cell or materialthat is associated with cancer.

The cancer antigen can be any type of cancer antigen known in the art.The cancer antigen may be an epithelial cancer antigen, (e.g., breast,gastrointestinal, lung), a prostate specific cancer antigen (PSA) orprostate specific membrane antigen (PSMA), a bladder cancer antigen, alung (e.g., small cell lung) cancer antigen, a colon cancer antigen, anovarian cancer antigen, a brain cancer antigen, a gastric cancerantigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, aliver cancer antigen, an esophageal cancer antigen, a head and neckcancer antigen, or a colorectal cancer antigen.

In another embodiment, the cancer antigen is a lymphoma antigen (e.g.,non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancerantigen, a leukemia antigen, a myeloma (i.e., multiple myeloma or plasmacell myeloma) antigen, an acute lymphoblastic leukemia antigen, achronic myeloid leukemia antigen, or an acute myelogenous leukemiaantigen.

In another embodiment, the cancer antigen is a mucin-1 protein orpeptide (MUC-1) that is found on all human adenocarcinomas: pancreas,colon, breast, ovarian, lung, prostate, head and neck, includingmultiple myelomas and some B cell lymphomas. Patients with inflammatorybowel disease, either Crohn's disease or ulcerative colitis, are at anincreased risk for developing colorectal carcinoma. MUC-1 is a type Itransmembrane glycoprotein. The major extracellular portion of MUC-1 hasa large number of tandem repeats consisting of 20 amino acids whichcomprise immunogenic epitopes. In some cancers it is exposed in anunglycosylated form that is recognized by the immune system. SeeGendler, S. J., et al., J. Biol. Chem. 265:15286-15293 (1990).

In another embodiment, the cancer antigen is a mutated B-Raf antigen,which is associated with melanoma and colon cancer. The vast majority ofthese mutations represent a single nucleotide change of T-A atnucleotide 1796 resulting in a valine to glutamic acid change at residue599 within the activation segment of B-Raf. Raf proteins are alsoindirectly associated with cancer as effectors of activated Rasproteins, oncogenic forms of which are present in approximatelyone-third of all human cancers. Normal non-mutated B-Raf is involved incell signaling, relaying signals from the cell membrane to the nucleus.The protein is usually only active when needed to relay signals. Incontrast, mutant B-Raf has been reported to be constantly active,disrupting the signaling relay. Mercer and Pritchard, Biochim BiophysActa. (2003);1653(1):25-40; Sharkey, et al, Cancer Res.(2004);64(5):1595-9.

In one embodiment, the cancer antigen is a human epidermal growth factorreceptor-2 (HER-2/neu) antigen. Cancers that have cells that overexpressHER-2/neu are referred to as HER-2/neu⁺ cancers. Exemplary HER-2/neu⁺cancers include prostate cancer, lung cancer, breast cancer, ovariancancer, pancreatic cancer, skin cancer, liver cancer (e.g.,hepatocellular adenocarcinoma), intestinal cancer, and bladder cancer.

HER-2/neu has an extracellular binding domain (ECD) of approximately 645aa, with 40% homology to epidermal growth factor receptor (EGFR), ahighly hydrophobic transmembrane anchor domain (TMD), and acarboxyterminal intracellular domain (ICD) of approximately 580 aa with80% homology to EGFR. The nucleotide sequence of HER-2/neu is availableat GENBANK® Accession Nos. AH002823 (human HER-2 gene, promoter regionand exon 1); M16792 (human HER-2 gene, exon 4): M16791 (human HER-2gene, exon 3); M16790 (human HER-2 gene, exon 2); and M16789 (humanHER-2 gene, promoter region and exon 1). The amino acid sequence for theHER-2/neu protein is available at GENBANK® Accession No. AAA58637. Basedon these sequences, one skilled in the art could develop HER-2/neuantigens using known assays to find appropriate epitopes that generatean effective immune response. Exemplary HER-2/neu antigens includep369-377 (a HER-2/neu derived HLA-A2 peptide); dHER2 (CorixaCorporation); li-Key MHC class II epitope hybrid (Generex BiotechnologyCorporation); peptide P4 (amino acids 378-398); peptide P7 (amino acids610-623); mixture of peptides P6 (amino acids 544-560) and P7; mixtureof peptides P4, P6 and P7; HER2 [9₇₅₄]; and the like.

In one embodiment, the cancer antigen is an epidermal growth factorreceptor (EGFR) antigen. The EGFR antigen can be an EGFR variant 1antigen, an EGFR variant 2 antigen, an EGFR variant 3 antigen and/or anEGFR variant 4 antigen. Cancers with cells that overexpress EGFR arereferred to as EGFR⁺ cancers. Exemplary EGFR⁺ cancers include lungcancer, head and neck cancer, colon cancer, colorectal cancer, breastcancer, prostate cancer, gastric cancer, ovarian cancer, brain cancerand bladder cancer.

The nucleotide sequence (mRNA) of EGFR variant 1 is available atGENBANK® Accession No. NM_(—)005228. The nucleotide sequence (mRNA) ofEGFR variant 2 is available at GENBANK® Accession No. NM_(—)201282. Thenucleotide sequence (mRNA) of EGFR variant 3 is available at GENBANK®Accession No. NM_(—)201283. The nucleotide sequence (mRNA) of EGFRvariant 4 is available at GENBANK® Accession No. NM_(—)201284. ExemplaryEGFR antigens include GI-3001; peptide aa 1168-1181; and the like.

In one embodiment, the cancer antigen is a vascular endothelial growthfactor receptor (VEGFR) antigen. The VEGFR antigen can be VEGFR variant1 antigen or VEGFR variant 2 antigen. VEGFR variant 1 is synonymous withFlt-1. VEGFR variant 2 is synonymous with Flk-1 and Kdr (i.e., kinaseinsert domain protein receptor). VEGFR is considered to be a regulatorof cancer-induced angiogenesis. Cancers with cells that overexpressVEGFR are called VEGFR⁺ cancers. Exemplary VEGFR⁺ cancers include breastcancer, lung cancer, small cell lung cancer, colon cancer, colorectalcancer, renal cancer, leukemia, and lymphocytic leukemia.

The nucleotide sequence (DNA) for VEGFR variant 1 (Flt-1) is availableat GENBANK® Accession No. D64016 (human gene for vascular endothelialgrowth factor receptor, promoter and exon 1). The nucleotide sequence(mRNA) for VEGFR variant 2 (Flk-1 or Kdr) is available at GENBANK®Accession No. AF063658 (human vascular endothelial growth factorreceptor 2).

In one embodiment the cancer antigen is prostate-specific antigen (PSA)and/or prostate-specific membrane antigen (PSMA) that are prevalentlyexpressed in androgen-independent prostate cancers.

In another embodiment, the cancer antigen is Gp-100 Glycoprotein 100 (gp100) is a tumor-specific antigen associated with melanoma.

In one embodiment, the cancer antigen is a carcinoembryonic (CEA)antigen. Cancers with cells that overexpress CEA are referred to as CEA⁺cancers. Exemplary CEA⁺ cancers include colorectal cancer, gastriccancer and pancreatic cancer. The nucleotide sequence (mRNA) for humancarcinoembryonic antigen-like 1 is available at GENBANK® Accession No.NM_(—)020219. Exemplary CEA antigens include CAP-1 (i.e., CEA aa571-579), CAP1-6D, CAP-2 (i.e., CEA aa 555-579), CAP-3 (i.e., CEA aa87-89), CAP-4 (CEA aa 1-11), CAP-5 (i.e., CEA aa 345-354), CAP-6 (i.e.,CEA aa 19-28) and CAP-7.

In one embodiment, the cancer antigen is carbohydrate antigen 10.9 (CA19.9). CA 19.9 is an oligosaccharide related to the Lewis A blood groupsubstance and is associated with colorectal cancers.

In another embodiment, the cancer antigen is a melanoma cancer antigen.Melanoma cancer antigens are useful for treating melanoma. Exemplarymelanoma cancer antigens include MART-1 (e.g., MART-1 26-35 peptide,MART-1 27-35 peptide); MART-1/Melan A; pMel17; pMel17/gp 100; gp100(e.g., gp 100 peptide 280-288, gp 100 peptide 154-162, gp 100 peptide457-467); TRP-1; TRP-2; NY-ESO-1; p16; beta-catenin; mum-1; and thelike.

In one embodiment, the cancer antigen is a mutant or wild type raspeptide. The mutant ras peptide can be a mutant K-ras peptide, a mutantN-ras peptide and/or a mutant H-ras peptide. Mutations in the rasprotein typically occur at positions 12 (e.g., arginine or valinesubstituted for glycine), 13 (e.g., asparagine for glycine), 61 (e.g.,glutamine to leucine) and/or 59. Mutant ras peptides can be useful aslung cancer antigens, gastrointestinal cancer antigens, hepatomaantigens, myeloid cancer antigens (e.g., acute leukemia,myelodysplasia), skin cancer antigens (e.g., melanoma, basal cell,squamous cell), bladder cancer antigens, colon cancer antigens,colorectal cancer antigens, and renal cell cancer antigens.

In another embodiment of the invention, the cancer antigen is a mutantand/or wildtype p53 peptide. The p53 peptide can be used as colon cancerantigens, lung cancer antigens, breast cancer antigens, hepatocellularcarcinoma cancer antigens, lymphoma cancer antigens, prostate cancerantigens, thyroid cancer antigens, bladder cancer antigens, pancreaticcancer antigens and ovarian cancer antigens.

The cancer antigen can be a cell, a protein, a peptide, a fusionprotein, DNA encoding a peptide or protein, RNA encoding a peptide orprotein, a glycoprotein, a lipoprotein, a phosphoprotein, acarbohydrate, a lipopolysaccharide, a lipid, a chemically linkedcombination of two or more thereof, a fusion or two or more thereof, ora mixture of two or more thereof. In another embodiment, the cancerantigen is a peptide comprising about 6 to about 24 amino acids; fromabout 8 to about 20 amino acids; from about 8 to about 12 amino acids;from about 8 to about 10 amino acids; or from about 12 to about 20 aminoacids. In one embodiment, the cancer antigen is a peptide having a MHCClass I binding motif or a MHC Class II binding motif. In anotherembodiment, the cancer antigen comprises a peptide that corresponds toone or more cytotoxic T lymphocyte (CTL) epitopes.

In another embodiment, the cancer antigen is in the form of a foreignhomologous cancer antigen. Foreign homologous cancer antigens andmethods for making them are described in U.S. Pat. No. 6,942,862.Because many human cancer antigens are self proteins (i.e., proteinsnormally produced by an individual and not necessarily unique tocancer), immunologic tolerance may exist and represent a barrier toeffective vaccination against the human cancer antigens. This aspect ofthe invention overcomes immunologic tolerance by immunizing a patientwith a protein or peptide that is foreign (i.e., not identical to thatin the individual) but nevertheless homologous to an individual's selfcancer antigen or portion thereof. “Foreign” cancer antigens can begenerated from, for example, rabbits, rats, mice and pigs. Generally, aforeign cancer antigen (e.g., protein or peptide) will possess at leastabout 75% sequence homology to the cancer antigen targeted. Sequencehomology means either identical amino acids at the same positions in thesequence (i.e., sequence identity), or conservative substitutions ofamino acids at the same positions in the sequence. Conservativesubstitutions are well known in the art. Examples are isoleucine forleucine, valine for alanine, glutamic acid for aspartic acid, threoninefor serine, etc. Typically, a foreign cancer antigen (e.g., proteins orpeptides) will possess about 80%, 85%, 90%, 95% or 99% sequencehomology. Preferred foreign cancer antigens (e.g., proteins or peptides)are those which are highly homologous, e.g., with from about but lessthan 100% sequence homology. Particularly preferred foreign cancerantigens (e.g., proteins or peptides) are those wherein theaforementioned sequence homology percents each represent percentsequence identity.

4.2 Viral Antigens Derived from Virus Associated with Cancer

One of the immunotherapeutic agents of the pharmaceutical compositionmay be one or more antigens derived from a virus associated with cancer.Infection from certain viruses are known to lead to the development ofdifferent types of cancers, for example, human papilloma virus (HPV),hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1) and human T-cellleukemia virus-2 (HTLV-2).

Patients who are infected or who are at risk of being infected with thehuman papilloma virus (HPV) are at a higher risk for developing cervicalcancer than HPV negative patients. The risk for cervical cancer isparticularly high for patients who have HPV-16, HPV-18, HPV-31, HPV-33and/or HPV-35 infections. The HPV antigen that can be used in thepharmaceutical compositions and the methods of the invention can be anHPV-16 antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigenand/or an HPV-35 antigen; and is preferably an HPV-16 antigen and/orHPV-18 antigen. The genome of HPV-16 is described in Virology,145:181-185 (1985) and DNA sequences encoding HPV-18 are described inU.S. Pat. No. 5,840,306, the disclosures of which are incorporated byreference herein in their entirety. HPV-16 antigens (e.g., seroreactiveregions of the E1 and/or E2 proteins of HPV-16) are described in U.S.Pat. No. 6,531,127, and HPV-18 antigens (e.g., seroreactive regions ofthe L1 and/or L2 proteins of HPV-18) are described in U.S. Pat. No.5,840,306, the disclosures of which are incorporated by referenceherein. Based on the sequences and antigens for HPV-16 and HPV-18described in these references, one skilled in the art could developother HPV antigens using known assays to find appropriate epitopes thatgenerate an effective immune response.

Patients who are infected or are at risk of being infected withhepatitis viral infections, such as hepatitis B (HBV) and/or hepatitis C(HCV) viral infections, are at a higher risk for developing liver cancerthan patients who do not have hepatitis viral infections. HBV antigensand HCV antigens can be used in the pharmaceutical compositions and themethods of the invention. The complete genome for HBV is available atGENBANK® Accession No. NC_(—)003977, the disclosure of which isincorporated herein. The genome of HCV is described in European PatentApplication No. 318 216, the disclosure of which is incorporated herein.PCT/US90/01348, incorporated by reference herein, discloses sequenceinformation of clones of the HCV genome, amino acid sequences of HCVviral proteins and methods of making and using such compositions for HCVvaccines comprising HCV proteins and peptides derived there from. Basedon the sequences and antigens for HBV and HCV described in thesereferences, one skilled in the art could develop other HBV and/or HCVantigens using known assays to find appropriate epitopes that generatean effective immune response.

Patients who are infected or are at risk for being infected withEpstein-Barr virus (EBV) are at a higher risk for developing Burkitt'slymphoma, nasopharyngeal carcinoma and Hodgkin's disease than EBVnegative patients. An EBV antigen can be used in the pharmaceuticalcompositions and the methods of the invention. The nucleotide sequenceof EBV DNA is described, for example, in U.S. Pat. No. 4,707,358. Basedon this sequences for EBV, one skilled in the art could develop EBVantigens using known assays to find appropriate epitopes that generatean effective immune response. The compounds of the invention, EBVantigens and immunostimulatory compounds can be administered separatelyor in the form of a composition. The composition can be in the form of aprophylactic vaccine (i.e., for patients who are EBV negative) ortherapeutic vaccine (i.e., for patients who are EBV positive).

Patients who are infected or are at risk of being infected with humanherpes virus 8 (HHV-8) are at a higher risk for developing Kaposi'ssarcoma than HHV-8 negative patients. The HHV-8 antigen can be used inthe pharmaceutical compositions and the methods of the invention. Thenucleotide sequence of HHV-8 is described, for example, by Russo et al,“Nucleotide sequence of the Kaposi sarcoma-associated herpes virus(HHV8),” Proc. Natl. Acad. Sci USA, 93:14862-14867 (1996). Based on theknown sequence for HHV-8, one skilled in the art could develop HHV-8antigens using known assays to find appropriate epitopes that generatean effective immune response.

Patients who are infected or at risk of being infected with human T-cellleukemia virus-1 (HTLV-1) or human T-cell leukemia virus-2 (HTLV-2) areat a higher risk for developing T-cell leukemia than HTLV-1 or HTLV-2negative patients. The sequences of HTLV-1 and HTLV-2 are well known inthe art and are described in Wong-Staal F, Gallo R C. HumanT-lymphotropic retroviruses. Nature 317:395-403, 1985.

4.3 Anti-Cancer Antibodies

One of the immunotherapeutic agents of the pharmaceutical compositionmay be one or more anti-cancer antibodies, that is, an antibody that hasbeen generated to one or more cancer antigens. Exemplary anti-cancerantibodies include the following:

trastuzumab (HERCEPTIN® by Genentech) which is used to treat HER-2/neupositive breast cancer or metastatic breast cancer;

bevacizumab (AVASTIN® by Genentech) which is used to treat colorectalcancer, metastatic colorectal cancer, breast cancer, metastatic breastcancer, non-small cell lung cancer, or renal cell carcinoma;

rituximab (RITUXAN® by Genentech), which is used to treat non-Hodgkin'slymphoma or chronic lymphocytic leukemia;

pertuzumab (OMNITARG® by Genentech) which is used to treat breastcancer, prostate cancer, non-small cell lung cancer, or ovarian cancer;

cetuximab (ERBITUX® by ImClone Systems Incorporated) which can be usedto treat colorectal cancer, metastatic colorectal cancer, lung cancer,head and neck cancer, colon cancer, breast cancer, prostate cancer,gastric cancer, ovarian cancer, brain cancer, pancreatic cancer,esophageal cancer, renal cell cancer, prostate cancer, cervical cancer,or bladder cancer;

IMC-1C11 (ImClone Systems Incorporated) which is used to treatcolorectal cancer, head and neck cancer, as well as other potentialcancer targets;

tositumomab and tositumomab and iodine I¹³¹ (BEXXAR® by CorixaCorporation) which is used to treat non-Hodgkin's lymphoma, which can beCD20 positive, follicular, non-Hodgkin's lymphoma, with and withouttransformation, whose disease is refractory to Rituximab and hasrelapsed following chemotherapy;

In¹¹¹ ibirtumomab tiuxetan; Y⁹⁰ ibirtumomab tiuxetan; In¹¹¹ ibirtumomabtiuxetan and Y⁹⁰ ibirtumomab tiuxetan (ZEVALIN® by Biogen Idec) which isused to treat lymphoma or non-Hodgkin's lymphoma, which can includerelapsed follicular lymphoma; relapsed or refractory, low grade orfollicular non-Hodgkin's lymphoma; or transformed B-cell non-Hodgkin'slymphoma;

EMD 7200 (EMD Pharmaceuticals) which is used for treating for treatingcancer is non-small cell lung cancer or cervical cancer;

SGN-30 (genetically engineered monoclonal antibody targeted to CD30antigen by Seattle Genetics) (Hodgkin's lymphoma or non-Hodgkin'slymphoma); SGN-15 (genetically engineered monoclonal antibody targetedto a Lewis^(γ)-related antigen that is conjugated to doxorubicin bySeattle Genetics) (non-small cell lung cancer); SGN-40 (humanizedmonoclonal antibody targeted to CD40 antigen by Seattle Genetics)(multiple myeloma or non-Hodgkin's lymphoma); SGN-35 (geneticallyengineered monoclonal antibody targeted to a CD30 antigen that isconjugated to Auristatin E by Seattle Genetics) (non-Hodgkin'slymphoma); SGN-17/19 (fusion protein containing antibody and enzymeconjugated to melphalan prodrug by Seattle Genetics) (melanoma ormetastatic melanoma).

The anti-cancer antibody can be a fragment of an antibody; a complexcomprising an antibody; or a conjugate comprising an antibody. Theantibody can optionally be chimeric or humanized.

The mechanism(s) of action of many of these antibodies are not entirelyclear, but generation of an immune response such as antibody-dependentcell-mediated cytotoxicity (ADCC) is often believed to be part of thetherapeutic action. Activation of ADCC by TLR4 ligation on Fcreceptor-bearing cells may enhance the anti-tumor efficacy of theantibody. The use of the anti-cancer antibodies in combination with oneor more compounds selected from formulae (I), (II), (III), (IV), and (V)will increase the immune response.

4.4 Anti-Idiotypic Antibodies

Antibodies to antigens have a serologically unique structure at theantigen-binding site, called an idiotype. An antibody may be generatedto the original antibody, resulting in the production of anti-idiotypicantibodies. The original antibody is designated Ab1, and theanti-idiotypic antibody Ab2. The Ab2 antibodies recognize theantigen-binding site of Ab1, and therefore share a motif or structuralsimilarities with the original antigen. An antibody raised to thebinding site on Ab2 may therefore react with the original antigen. Ifthe original antigen is a cancer antigen, the anti-Ab2 antibody may havea therapeutic effect.

When administered alone, the anti-idiotypic antibody may not be able togenerate sufficient immune response to a cancer antigen. However, whenthe anti-idiotypic antibody is used in combination with one or morecompound selected from formulae (I), (II), (III), (IV), and (V) as apharmaceutical composition or in a method of administration of thisinvention, an immune response is generated. The adjuvant compounds offormulae (I), (II), (III), (IV), and (V) can improve the immunogenicityof the anti-idiotypic antibody or provide the anti-idiotypic antibodywith the ability to generate an immune response by breakingimmunological tolerance. In addition, the pharmaceutical composition ofthe invention may also reduce the amount of anti-idiotypic antibodiesneeded to induce an immune response and/or reduce the number ofadministrations needed to induce the desired immune response.

Anti-idiotypic antibodies are known in the art. The anti-idiotypicantibody may be an antibody to an antibody that that is produced inresponse to a cancer antigen described above or to an anti-cancerantibody. Exemplary anti-idiotypic antibodies include 105AD7 (describedin U.S. Pat. No. 6,042,827, the disclosure of which is incorporated byreference herein in its entirety); BEC2 (ImClone Systems Incorporated);IGN301 (Igeneon, a subsidiary of Aphton Corporation); and the like. Theproduction of anti-idiotypic antibodies are well known in the art andare described for example in U.S. Pat. No. 6,926,893, the disclosure ofwhich is incorporated by reference herein in its entirety.

4.5 Modes of Administration of Pharmaceutical Compositions

Administration of the pharmaceutical composition can be accomplished byseveral routes that would be suitable for delivery. Exemplary deliverymodes include parenteral administration, e.g., subcutaneous injection,transcutaneous, intravenous, intra-tumoral, peri-tumoral, intra-nasal,ophthalmic, intramuscular, intradermal, intraperitoneal, pulmonary, andnon-parenteral administration, e.g., transmucosal, transdermal,inhalation, intravaginal, rectal or oral administration.

4.6 Methods of Treatment

The pharmaceutical composition provides a method for stimulating oreliciting or enhancing an immune response in a subject individual. Thesubject individual is preferably human, although the invention can beapplied in veterinary applications to animal species, including mammalsor avian species.

The subject individual may be at risk for developing cancer, diagnosedwith a cancer, in treatment for cancer, or in post-therapy recovery fromcancer.

The term “immune response” encompasses both cellular and humoral immuneresponses, including stimulating the production of cytokines,stimulating the proliferation of immune cells, stimulating theactivation of immune cells, or stimulating the lytic activity of immunecells. Examples of immune responses stimulated by the methods of theinvention are the secretion of cytokines, the activation of NK cells,the proliferation of B cells, T cells, macrophages, monocytes, and otherimmune cells, and other immune responses. To detect a cellular immuneresponse, for example, T cell effector activity against cells expressingthe antigen can be detected using standard assays, e.g., target-cellkilling, macrophage activation, B-cell activation or lymphokineproduction. Humoral responses can be measured by detecting theappearance of, or the increase in titer of, for example,antigen-specific antibodies, using routine methods such as ELISA. Theprogress of the antibody response can be determined by measuring classswitching, such as the switch from an early IgM response to a later IgGresponse.

As used herein, the term “stimulate an immune response” includesstimulating, eliciting, increasing, enhancing, sustaining, and/orimproving the stimulation of new immune response or of a preexistingimmune response. Thus, “stimulating an immune response” as animmunotherapy refers to enhancing the therapeutic efficacy, increasingsurvival time, slowing the progression of a cancerous tumor or shrinkingthe cancerous tumor size, preventing the spread of a tumor or ofmetastases, preventing or slowing the recurrence of treated cancer,eliminating cancer cells not killed by earlier treatments, targetingpotential cancer cells or targeting antigens derived from a virusassociated with cancer. In the methods of this invention, theimmunotherapeutic agent and the compound of selected from formulae (I),(II), (III), (IV) and (V) are administered in an amount effective tostimulate an immune response in the subject individual at a dosesufficient to generate an effective immune response without unacceptabletoxicity. As will be understood by one of skill in the art, themagnitude of the immune response and the maintenance of that responsemay have varying degrees which will be recognized a having a potentialtherapeutic or prophylactic benefit.

To stimulate an immune response, the subject individual is administered(i) at least one immunotherapeutic agent selected from one or morecancer antigens, one or more antigens derived from a virus associatedwith cancer, an anti-cancer antibody, and an anti-idiotypic antibody toan anti-cancer antibody, and (ii) one or more compounds selected fromformulae (I), (II), (III), (IV) and (V), and/or a pharmaceuticallyacceptable salt, hydrate, solvate, stereoisomer, amorphous solidthereof, or any combination thereof. Typically, the administration ofthe immunotherapeutic agent and the compound of selected from formulae(I), (II), (III), (IV) and (V) will be in the form of a vaccine oradministered in a vaccine regimen. The therapeutic agent and thecompound of selected from formulae (I), (II), (III), (IV) and (V) can beadministered at about the same time to the subject individual, or can beadministered separately and/or sequentially. “At about the same time”includes administering one or more immunotherapeutic agents and one ormore compounds selected from formulae (I), (II), (III), (IV), and (V) atthe same time, at the same time, but through different modes ofadministration or at different sites on the body, at different times onthe same day, or on different days, provided that they are administeredas part of an overall dosing treatment regimen. When administeredseparately, or sequentially, the one or more immunotherapeutic agentsand one or more compounds selected from formulae (I), (II), (III), (IV),and (V) are part of an overall treatment regimen, such as a therapeuticcocktail or a combination therapy. The dosing schedule can be routinelydetermined by one of skill in the art and may be varied or modifiedaccording to the appropriate treatment for the subject individual. Forexample, the immunotherapeutic agent and a compound of formulae (I),(II), (III), (IV) and (V) can be administered at about the same time asa single dose, or by a dose of the therapeutic agent and a dose of acompound of formulae (I), (II), (III), (IV) and (V). The dosing schedulecan be continued at regular intervals, such as at 1 to 4 week timeperiods, followed by dosing at regular intervals of 1 to 3 months, forexample. In another embodiment, the dosing schedule can be based on a“prime” and “boost” treatment, in which the immunotherapeutic agent isadministered to prime or stimulate the production of CTLs and thenanother dose of immunotherapeutic agent in combination with one or morecompounds selected from formulae (I), (II), (III), (IV), and (V) toboost the production of neutralizing antibodies and antibody dependentcellular cytotoxity. The immune response in the subject individual canbe assessed and monitored through known methods.

In some instance, these treatments can be used in combination withconventional cancer therapies or pharmaceutical formulations useful fortreating cancer or infectious diseases. These treatments can includesurgical procedures, radiation therapy and/or ablation therapy (e.g.,laser therapy, infrared therapy and the like).

Cancer therapies including dendritic cell therapy, chemokines,cytokines, tumor necrosis factors (e.g., TNF-α), chemotherapeutic agents(e.g., adenosine analogs (e.g., cladribine, pentostatin), alkylsulfanates (e.g., busulfan)), anti-tumoral antibiotics (e.g., bleomycin,dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin,mitoxantrone, mitomycin), aziridines (e.g., thiotepa), camptothecinanalogs (e.g., irinotecan, topotecan), cryptophycins (e.g., cryptophycin52, cryptophicin 1), dolastatins (e.g., dolastatin 10, dolastatin 15),enedyine anticancer drugs (e.g., esperamicin, calicheamicin, dynemicin,neocarzinostatin, neocarzinostatin chromophore, kedarcidin, kedarcidinchromophore, C-1027 chromophore, and the like), epipodophyllotoxins(e.g., etoposide, teniposide), folate analogs (e.g., methotrexate),maytansinoids (e.g., maytansinol and maytansinol analogues), microtubuleagents (e.g., docetaxel, paclitaxel, vinblastine, vincristine,vinorelbine), nitrogen mustards (e.g., chlorambucil, cyclophosphamide,estramustine, ifosfamide, mechlorethamine, melphalan), nitrosoureas(e.g., carmustine, lamustine, streptoxacin), nonclassic alkylators(e.g., altretamine, dacarbazine, procarbazine, temozolamide), platinumcomplexes (e.g., carboplatin, cisplatin), purine analogs (e.g.,fludarabine, mercaptopurine, thioguanine), pyrimidine analogs (e.g.,capecitabine, cytarabine, depocyt, floxuridine, fluorouracil,gemcitabine), substituted ureas (e.g., hydroxyurea)]; anti-angiogenicagents (e.g., canstatin, troponin I,), biologic agents (e.g., ZD 1839,virulizin and interferon), antibodies and fragments thereof (e.g., antiEGFR, anti-HER-2/neu, anti-KDR, IMC-C225), anti-emetics (e.g.,lorazepam, metoclopramide, and domperidone), epithelial growth factorinhibitors (e.g., transforming growth factor beta 1), anti-mucositicagents (e.g., dyclonine, lignocaine, azelastine, glutamine, corticoidsteroids and allopurinol), anti-osteoclastic agents (e.g.,bisphosphonates {e.g., etidronate, pamidronate, ibandronate, andosteoprotegerin}), hormone regulating agents (e.g., anti-androgens, LHRHagonists, anastrozole, tamoxifen), hematopoietic growth factors,anti-toxicity agents (e.g., amifostine), kinase inhibitors (gefitinib,imatinib), and mixtures of two or more thereof.

Antibodies that block immunosuppressive functions, for example,anti-CTLA4 antibodies that block a receptor on T cells that turns offactivation may also be used in combination with the immunotherapeuticagent and a compound of formulae (I), (II), (III), (IV) and (V). Thus,administering the immunotherapeutic agent and a compound of formulae(I), (II), (III), (IV) and (V) with anti-CTLA4 antibodies will increaseimmune response in the subject individual.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers to the study of how apatient's genes determine his or her response to a drug (e.g., apatient's “drug response phenotype” or “drug response genotype”). Thus,another aspect of the invention provides methods for tailoring anindividual's prophylactic or therapeutic treatment according to thatindividual's drug response genotype. Pharmacogenomics allows a clinicianor physician to target prophylactic or therapeutic treatments topatients who will most benefit from the treatment and to avoid treatmentof patients who will experience toxic drug-related side effects. Theclinician or physician can thereby tailor the type of treatment that maybe necessary to the specific patient.

4.7 Optional Immunostimulatory Compounds

In one embodiment of the methods of immunotherapy, the immune responseis further augmented by the administration of compounds that may act asan immunostimulatory compound. Exemplary immunostimulatory compoundsinclude toll like receptor (TLR) agonists (e.g., TLR4, TLR7, TLR9),N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), lipopolysaccharides(LPS), genetically modified and/or degraded LPS, alum, glucan, colonystimulating factors (e.g., EPO, GM-CSF, G-CSF, M-CSF, pegylated G-CSF,SCF, IL-3, IL6, PIXY 321), interferons (e.g., γ-interferon,α-interferon), interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18), MHCClass II binding peptides, saponins (e.g., QS21), unmethylated CpGsequences, 1-methyl tryptophan, arginase inhibitors, cyclophosphamide,antibodies that block immunosuppressive functions (e.g., anti-CTLA4antibodies), and mixtures of two or more thereof. Exemplary TLR4agonists include lipopolysaccharides (LPS); E. coli LPS; and P.gingivalis LPS. Exemplary TLR7 agonists include imidazoquinolinecompounds (e.g., imiquimod, resiquimod and the like); and loxoribine.

4.8 Pharmaceutical Formulations

The pharmaceutical composition is formulated to be compatible with itsintended route of administration, and will typically include apharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions. The pharmaceutical compositions may be formulatedaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, (eds. J. Swarbrick and J. C. Boylan), 1988-1999, MarcelDekker, New York).

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the selectedparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride are included in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating compoundselected from formulae (I), (II), (III), (IV), and (V) in the specifiedamount in an appropriate solvent with one or a combination ofingredients enumerated above, as needed, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and other ingredients selected from those enumeratedabove or others known in the art. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationcan be vacuum drying and freeze-drying, which yields a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

4.9 Covalent Bonding, Complexes and Conjugates

In one embodiment, one or more immunotherapeutic agent and a compoundselected from formulae (I), (II), (III), (IV), and (V) can be covalentlybonded together through an alkyl, amino, carbonyl, ether, hydroxyl,phosphate, phosphonyl, sulfonyl, sulfate, thiol ether, or thiol moietyof compound selected from formulae (I), (II), (III), (IV), and (V). Forexample, one or more immunotherapeutic agent can be covalently bonded tothe R¹, X¹ and/or Y¹ substituents in the compounds selected fromformulae (I), (II), (III), (IV), and (V). For example, theimmunotherapeutic agent can be covalently bonded to a carbonyl moiety(e.g., the C₁ carbonyl) of the —C(O)— group or to the—C(O)—C₁₋₁₄alkyl-C(O)— group of the R¹ substituent in one or morecompounds of the invention. As another example, the immunotherapeuticagent can be covalently bonded through a nitrogen atom in the X¹ and/orY¹ substituents in the compound selected from formulae (I), (II), (III),(IV), and (V). One skilled in the art would be able to link one or moreimmunotherapeutic agent to a compound selected from formulae (I), (II),(III), (IV), and (V) following the methods described, for example, byHoffman et al., Biol. Chem. Hoppe-Sayler, 370:575-582 (1989); Wiesmulleret al., Vaccine, 7:29-33 (1989); Wiesmuller et al., Int. J. PeptideProtein Res., 40:255-260 (1992); Defourt et al., Proc. Natl. Acad. Sci.,89:3879-3883 (1992); Tohokuni et al., J. Am. Chem. Soc., 116:395-396(1994); Reichel, Chem. Commun., 2087-2088 (1997); Kamitakahara, Agnew.Chem. Int. Ed. 37:1524-1528 (1998); Dullenkopf et al., Chem. Eur. J.,5:2432-2438 (1999); the disclosures of which are incorporated byreference herein in their entirety.

In one embodiment, the immunotherapeutic agent can be in the form of acomplex. The complex can comprise at least one cancer antigen(optionally bonded to the compound selected from formulae (I), (II),(III), (IV), and (V)) and one or more proteins, peptides,immunostimulatory compounds and/or cells. Exemplary proteins andpeptides include heat shock proteins, heat shock peptides, MHC Class Iproteins, MHC Class I peptides, MHC Class II proteins, MHC Class IIpeptides, and the like. Exemplary cells include dendritic cells,autologous dendritic cells, dendritic cells pulsed with cancer antigens,autologous dendritic cells pulsed with cancer antigens, and the like.Exemplary immunostimulatory compounds include TLR agonists (e.g., TLR4,TLR7, TLR9), N-acetylmuramyl-L-alanine-D-isoglutamine (MDP),lipopolysaccharides (LPS), genetically modified and/or degraded LPS,alum, glucan, colony stimulating factors (e.g., EPO, GM-CSF, G-CSF,M-CSF, PEGylated G-CSF, SCF, IL-3, IL6, PIXY 321), interferons (e.g.,γ-interferon, α-interferon), interleukins (e.g., IL-2, IL-7, IL-12,IL-15, IL-18), saponins (e.g., QS21), monophosphoryl lipid A, 3De-O-acylated monophosphoryl lipid A (3D-MPL), unmethylated CpGsequences, 1-methyl tryptophan, arginase inhibitors, cyclophosphamide,antibodies that block immunosuppressive functions (e.g., anti-CTLA4antibodies), and the like. The complex with at least one cancer antigen(optionally bonded to compound selected from formulae (I), (II), (III),(IV), and (V)) and the proteins, peptides, immunostimulatory compoundsand/or cells can be non-covalent, ionic, covalent, van der Waals'forces, hydrogen bonding, and the like.

In another embodiment, the cancer antigen (optionally bonded to compoundselected from formulae (I), (II), (III), (IV), and (V)) is in the formof a complex comprising a heat shock protein or heat shock peptide(HSP), particularly gp96. In one embodiment, the complex is formed by invitro peptide pulsing the heat shock proteins and/or heat shockpeptides. The complex can be covalent or non-covalent. In anotherembodiment, the cancer antigen (optionally bonded to compound selectedfrom formulae (I), (II), (III), (IV), and (V)) is in the form of acomplex comprising a peptide, at least one heat shock protein and/orheat shock peptide, and at least one MHC Class I and/or II protein orpeptide.

In another embodiment, the cancer antigen (optionally bonded to compoundselected from formulae (I), (II), (III), (IV), and (V)) is in the formof a complex comprising a peptide and dendritic cells. In oneembodiment, the complex is formed by in vitro peptide pulsing thedendritic cells to form cancer antigen loaded dendritic cells. Thedendritic cells can be pulsed with cancer antigens and/or organisms(e.g., recombinant viruses or bacteria) expressing cancer antigens. Thedendritic cells can be autologous.

In another embodiment, the cancer antigen (optionally bonded to compoundselected from formulae (I), (II), (III), (IV), and (V)) is in the formof a complex comprising a peptide and an MHC Class I protein or peptide;and/or comprising a peptide and a MHC Class II protein or peptide. Inone embodiment, the MHC Class I protein or peptide is HLA (e.g.,HLA-A*0201, HLA-A2, HLA-A3, HLA-A*1101, HLA-A*3101, HLA-A*3301,HLA-A*6801, HLA-A24). The complex can be covalent or non-covalent. Inanother embodiment, the cancer antigen (optionally bonded to compoundselected from formulae (I), (II), (III), (IV), and (V)) is in the formof a complex comprising a peptide, a MHC Class I and/or II protein orpeptide, and at least one heat shock protein and/or heat shock peptide.

In another embodiment, the cancer antigen is in the form of a conjugate.For example, one or more cancer antigens can be chemically linked to oneor more proteins, peptides, carbohydrates, polymers, lipids and/or toxicmoieties. The conjugate can be of the formula: A-L-X, wherein “A” is oneor more cancer antigens as described herein, “L” is one or more linkinggroups, and “X” is one or more proteins, peptide, carbohydrates,polymers, lipids and/or toxic moieties. Exemplary linking groups includechemical linking groups and peptide linking groups. The most commonmethods for linking “A” and “X” rely on the presence of free amino(α-amino or Lys), sulfhydryl (Cys), or carboxylic acid groups (Asp, Glu,or α-carboxyl). Linking methods should be used that link the peptides tothe carrier proteins, peptides, carbohydrates, lipids and/or toxicmoieties via the carboxy- or amino-terminal residue. Other commonlinking methods include maleimide and carbodiimide coupling chemistry.The linking group can be selected so that enzymes act upon the linkinggroup so that “A” and “X” separate from each other in vivo.Alternatively, a linking group can be selected so that “A” and “X”remain covalently bonded via the linking group in vivo. In still otherembodiments, “L” can simply be a covalent bond between “A” and “X.” “X”can be a peptide, protein (such as an MHC Class I protein or an MHCClass II protein); keyhole limpet hemocyanin; albumin; bovine serumalbumin; ovalbumin; rabbit serum albumin, antibody and the like. “X” canbe a peptide. The peptide can be cancer antigen that is the same as ordifferent from “A.” Such a conjugate may be referred to as a multipleantigen peptide (MAP), and the peptides can form a fusion protein withor without the presence of a linking group “L.” The peptide can be aClass I and/or Class II peptide that can assist in generating an immuneresponse. One skilled in the art will appreciate that the term “fusionprotein” may be used when two peptides are linked together, in whichcase the “L” group may drop out of the structure. In other embodiments,“X” can be a toxic moiety. Exemplary toxic moieties include viraltoxins, bacterial toxins (e.g., diphtheria toxins, tetanus toxins,clostridia toxin, cholera toxin, anthrax toxin, botulinum toxin,pertussis toxin, tracheal toxin,

5. COMPOUNDS OF FORMULAE (I), (II), (III), (IV) and (V)

The pharmaceutical compositions include the immunotherapeutic agentsdescribed above and a compound selected from formulae (I), (II), (III),(IV), and (V) and/or a pharmaceutically acceptable salt, hydrate,solvate, stereoisomer, amorphous solid thereof, or any combinationthereof.). The compounds of Formula (I), (II), (III), (IV) and (V) canfunction as adjuvants and/or as immunostimulatory compounds depending onthe application in which they are used.

wherein:

-   R¹ is:    -   (a) —C(O)—;    -   (b) —C(O)—C₁₋₁₄alkyl-C(O)— or —C(O)—C₁₋₁₄alkenyl-C(O)—;        -   wherein the —C₁₋₁₄alkyl- or —C₁₋₁₄alkenyl- is optionally            substituted with one or more substituents selected from            hydroxy, C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkyldioxy, C₁₋₅            alkylamino, carboxy, C₁₋₆ alkoxycarbonyl, C₁₋₆ carbamoyl,            C₁₋₆ acylamino, and/or (aryl)C₁₋₆alkyl; and            -   wherein the aryl moiety of the (aryl)C₁₋₆-alkyl is                optionally substituted with one or more substituents                selected from C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylamino,                C₁₋₆alkoxyamino, C₁₋₆alkylamino-C₁₋₆alkoxy,                —O—C₁₋₆alkylamino-C₁₋₆alkoxy,                —O—C₁₋₆alkylamino-C(O)—C₁₋₆alkyl-C(O)OH,                —O—C₁₋₆alkylamino-C(O)—C₁₋₆alkyl-C(O)—C₁₋₆alkyl,                —O—C₁₋₆alkyl-NH—C₁₋₆alkyl-O—C₁₋₆ alkyl,                —O—C₁₋₆alkyl-NH—C(O)—C₁₋₆alkyl-C(O)OH, and/or                —O—C₁₋₆alkyl-NH—C(O)—C₁₋₆alkyl-C(O)—C₁₋₆alkyl;    -   (c) a C₂ to C₁₅ straight or branched chain alkyl group        optionally substituted with one or more hydroxy and/or alkoxy        groups; or    -   (d) —C(O)—C₆₋₁₂aryl-C(O)— wherein the aryl is optionally        substituted with one or more hydroxy, halo (e.g., fluoro),        nitro, amino, C₁₋₆alkyl and/or C₁₋₆alkoxy groups;-   a and b are each independently 0, 1, 2, 3 or 4; (preferably 2);-   a′ and b′ are independently 2, 3, 4, 5, 6, 7 or 8; (preferably 2);-   d and e are each independently 1, 2, 3, 4, 5 or 6;-   d′ and e′ are each independently 0, 1, 2, 3 or 4; (preferably 0, 1    or 2);-   d″ and e″ are each independently 0, 1, 2, 3 or 4; (preferably 1, 2,    3 or 4);-   T is oxygen or sulfur;-   X¹, X², Y¹ and Y² are each independently null, oxygen, NH,    —N(C(O)C₁₋₄alkyl)-, or —N(C₁₋₄alkyl)-;-   W¹ and W² are each independently carbonyl, methylene, sulfone or    sulfoxide;-   R², R³, R⁴, R⁵, R⁶ and R⁷ are each independently:    -   (a) C₂ to C₂₀ straight chain or branched chain alkyl, which is        optionally substituted with one or more oxo, halo (preferably        fluoro), hydroxy and/or alkoxy groups;    -   (b) C₂ to C₂₀ straight chain or branched chain alkenyl, which is        optionally substituted with one or more of oxo, halo (preferably        fluoro), hydroxy and/or alkoxy groups;    -   (c) C₂ to C₂₀ straight chain or branched chain alkoxy, which is        optionally substituted with one or more oxo, halo (e.g.,        fluoro), hydroxy and/or alkoxy groups;    -   (d) —NH—C₂₋₂₀ straight chain or branched chain alkyl, wherein        the alkyl group is optionally substituted with one or more oxo,        halo (e.g., fluoro), hydroxy and/or alkoxy groups;    -   (e) —C(O)—C₂₋₂₀ straight chain or branched chain alkyl or        alkenyl, wherein the alkyl or alkenyl is optionally substituted        with one or more oxo, halo (e.g., fluoro), hydroxy and/or alkoxy        groups;    -   (f)

-   -    Z is O or NH; and M and N are each independently C₂ to C₂₀        straight chain or branched chain alkyl, alkenyl, alkoxy,        acyloxy, alkylamino, or acylamino;    -   (g)

-   -   -   R⁸ is C₁₋₆ straight or branched chain alkyl or C₂₋₆ straight            or branched chain alkenyl or alkynyl;        -   R⁹ and R¹⁰ are independently selected from the group            consisting of            -   (i) C₁ to C₂₀ straight chain or branched chain alkyl,                which is optionally substituted with one or more halo,                oxo, hydroxy and/or alkoxy; and            -   (ii) C₂ to C₂₀ straight chain or branched chain alkenyl                or alkynyl which is optionally substituted with one or                more halo, oxo, hydroxy and/or alkoxy;

-   G¹, G², G³, G⁴ and G⁵ are each independently oxygen, methylene,    —NH—, thiol, —N(C₁₋₄alkyl)-, —N[C(O)—C₁₋₄alkyl]-, —NH—C(O)—,    —NH—SO₂—, —C(O)—O—, —C(O)—NH—, —O—C(O)—, —O—C(O)—NH—, —O—C(O)—O—,    —NH—C(O)—NH—, —C(O)NH—, —C(O)N(C₁₋₄alkyl), aryl, and —S(O)_(n)—,    where n is 0, 1, or 2;

-   or G¹R², G²R⁴, G³R⁵ and/or G⁴R⁷ may together be a hydrogen atom or    hydroxyl;

-   Z¹ and Z² are each independently selected from —OP(O)(OH)₂,    —P(O)(OH)₂, —OP(O)(OR⁸)(OH) {where R⁸ is a C₁₋₄alkyl}, —OS(O)₂OH,    —S(O)₂OH—, —CO₂H, —OB(OH)₂, —OH, —CH₃, —NH₂, and —N(R⁹)₂ {where R⁹    is a C₁₋₄alkyl};

-   R¹² is H or a C₁₋₄ straight or branched alkyl; and

-   M is independently selected from a hydrogen atom and a    pharmaceutically acceptable cation {a monovalent cation will take    the place of one M, while a divalent cation will take the place of    two M variables}.

In one embodiment, R¹ in the compounds of Formula (I)-(V) is —C(O)— or—C(O)—C₁₋₁₄alkyl-C(O)—. In another embodiment, R¹ in the compounds ofFormula (I)-(V) is —C(O)—.

In one embodiment of the invention, T is oxygen in the compounds ofFormula (I)-(V).

In another embodiment, G¹, G², G³ and G⁴ in the compounds of Formula(I)-(V) are each independently oxygen, —NH—, —NH—C(O)—, —C(O)—O—,—C(O)—NH—, —O—C(O)—, —O—C(O)—NH—, —O—C(O)—O—, —NH—C(O)—NH—, or —C(O)NH—.In another embodiment, G¹, G², G³ and G⁴ in the compounds of Formula(I)-(V) are each independently oxygen, —C(O)—O— or —O—C(O)—. In anotherembodiment, G¹ and G³ in the compounds of Formula (I)-(V) are —O—C(O)—.

In one embodiment for the compounds of Formula (I)-(V) {preferablycompounds of Formula (I)-(III)}, R² and R⁵ are each independentlysubstituents selected from (a), (b), (c), (d) and (f) in the definitionsof R² and R⁵ herein; R³ and R⁶ are each independently substituentsselected from (a) and (b) in the definitions of R³ and R⁶ herein; and R⁴and R⁷ are each independently substituents selected from (a), (b), (c)and (e) in the definitions of R⁴ and R⁷ herein.

In other embodiments for the compounds of Formula (I)-(V) {preferablycompounds of Formula (IV) or (V)}, R², R³, R⁴, R⁵, R⁶ and R⁷ are eachindependently substituents selected from (a), (b), (g) and (h) in thedefinitions of R², R³, R⁴, R⁵, R⁶ and R⁷ herein.

In other embodiments for the compounds of Formulas (I)-(III), one ormore of the following is present: each of a and b is 2; each of X¹ andY¹ is NH; R¹ is —C(O)— or —C(O)—C₁₋₁₄alkyl-C(O)—; each of d′ and e′ is1; each of d″ and e″ is 1; X is O or NH, more preferably NH; and W isC(O); or each of d′ and e′ are 2.

In other embodiments for Formulas (I)-(III), R¹ is a —C(O)C₁₋₁₄alkyl-C(O)—, wherein the C₁₋₁₄alkyl is substituted, for example, with aC₁₋₅alkoxy group.

In one embodiment, the compounds of Formulas (I)-(III) are “Type 1”wherein the values of a and b are the same; the values of d and e arethe same; the values of d′ and e′ are the same; the values of d″ and e″are the same; X¹ and Y¹ are the same; X² and Y² are the same; W¹ and W²are the same; R² and R⁵ are the same; G¹ and G³ are the same; R³ and R⁶are the same; G² and G⁴ are the same; and R⁴ and R⁷ are the same.

In another embodiment, the compounds of Formulas (I)-(III) are “Type 2”wherein the values of a and b are different, the values of d and e arethe same, the values of d′ and e′ are different; the values of d″ and e″are the same; X¹ and Y¹ are different; X² and Y² are different; W¹ andW² are different; R² and R⁵ are different; G¹ and G³ are different; R³and R⁶ are different; G² and G⁴ are different; or R⁴ and R⁷ aredifferent.

In another embodiment, the compounds of Formulas (I)-(III) are “Type 3”wherein the values of a and b are different, the values of d and e aredifferent, the values of d′ and e′ are different; the values of d″ ande″ are different; X¹ and Y¹ are different; X² and Y² are different; W¹and W² are different; R² and R⁵ are different; G¹ and G³ are different;R³ and R⁶ are different; G² and G⁴ are different; or R⁴ and R⁷ aredifferent.

In other embodiments, the compounds of Formulas (I), (II) and/or (III)are preferably:

ER 803022; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 803058; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 803732; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804053; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804058; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804059; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804442; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804680; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

ER 804764; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

In one embodiment, the preferred compound is 112066; a stereoisomerthereof; a pharmaceutically acceptable salt thereof other than thesodium salt shown below; and/or the one or more sodium cations shownbelow can be replaced with hydrogen atoms:

In one embodiment, the preferred compound is ER 804057; apharmaceutically acceptable salt thereof other than the sodium saltshown below; or the one or more sodium cations shown below can bereplaced with hydrogen atoms:

In some embodiments for Formulas (IV) and (V), one or more of thefollowing limitations is present: each of a and b is 2; each of X¹ andY¹ is NH; each of d and e is 1 or 2; and each of d′ and e′ is 0, 1, or2. In certain preferred embodiments, each of d and e is 1 and each of d′and e′ is 0. In certain other preferred embodiments, each of d and e is1 and each of d′ and e′ is 1 or 2.

In some embodiments for Formulas (IV) and (V), R¹ is —C(O)— or—C(O)—C₁₋₁₄alkyl-C(O)—, wherein the C₁₋₁₄alkyl is optionally substitutedwith one or two substituents selected from the group consisting ofhydroxy, C₁₋₆alkoxy, C₁₋₆alkyldioxy, C₁₋₆alkylamino, or (aryl)C₁₋₆alkyl,wherein the aryl moiety of the (aryl)C₁₋₆alkyl is optionally substitutedwith C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkylamino, (C₁₋₆alkoxy)C₁₋₆alkylamino,(C₁₋₆alkylamino)C₁₋₆alkoxy, —O—C₁₋₆alkyl-NH—C₁₋₆alkyl-O—C₁₋₆alkyl,—O—C₁₋₆alkyl-NH—C(O)—C₁₋₆alkyl-C(O)OH, or—O—C₁₋₆alkyl-NH—C(O)—C₁₋₆alkyl-C(O)—C₁₋₆alkyl.

In some embodiments for Formulas (IV) and (V), G¹, G², G³, and G⁴ areeach independently selected from the group consisting of —NH—C(O)— and—O—C(O)—.

In some embodiments for Formula (IV) and (V), at least two of R², R³,R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₆₋₂₀ straight or branched chain alkyl,alkenyl, or alkynyl; any of which may optionally be substituted with oneor more substituents selected from the group consisting of halo, oxo,hydroxy and/or alkoxy. In other embodiments, at least two of R², R³, R⁴,R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₈₋₁₅ straight or branched chain alkyl,alkenyl, or alkynyl; any of which may optionally be substituted with oneor more substituents selected from the group consisting of halo, oxo,hydroxy and alkoxy.

In some embodiments for Formulas (IV) and (V), at least four of R², R³,R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₆₋₂₀ straight or branched chain alkyl,alkenyl or alkynyl; any of which may optionally be substituted with oneor more substituents selected from the group consisting of halo, oxo,hydroxy and alkoxy. In certain preferred embodiments, at least four ofR², R³, R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₈₋₁₅ straight or branched chainalkyl, alkenyl or alkynyl; any of which may optionally be substitutedwith one or more substituents selected from the group consisting ofhalo, oxo, hydroxy and alkoxy.

In some embodiments for Formulas (IV) and (V), at least six of R², R³,R⁴, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₆₋₂₀ straight or branched chain alkyl,alkenyl, or alkynyl; any of which may optionally be substituted with oneor more substituents selected from the group consisting of halo, oxo,hydroxy and alkoxy. In other embodiments, at least six of R², R³, R⁴,R⁵, R⁶, R⁷, R⁹ and R¹⁰ are C₈₋₁₅ straight or branched chain alkyl,alkenyl or alkynyl; any of which may optionally be substituted with oneor more substituents selected from the group consisting of halo, oxo,hydroxy and alkoxy.

In other embodiments, the invention provides compounds of Formula (I),(II), (III), (IV) or (V) wherein T is sulfur. In other embodiments, theinvention provides compounds of Formula (I), (II), (III), (IV) or (V)wherein T is sulfur; provided that the compound is not Compound No.804678.

In another embodiment of the invention, there is a proviso that thecompounds of Formula (I), (II) or (III) are not:

As used herein, the term “alkyl” includes substituted or unsubstituted,straight or branched chain monovalent or bivalent aliphatic hydrocarbongroups. One skilled in the art will appreciate the distinction between amonovalent alkyl group and a bivalent alkyl group in view of the contextof the term “alkyl” in the definition for any particular substituent.When an alkyl is a terminal group, it will be monovalent, such as —CH₃,—CH₂CH₃, —CH₂CH₂CH₃, —CH₂CH₂CH₂CH₃, and the like. When an alkyl isbetween other moieties, such as “—C(O)—C₁₋₁₄alkyl-C(O)—” in thedefinition of R¹, the alkyl group will be bivalent, such as —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, and the like.

As used herein, the term “alkenyl” includes substituted orunsubstituted, straight or branched chain unsaturated monovalent orbivalent aliphatic hydrocarbon groups. The “alkenyl” group can have anynumber of carbon-carbon double bonds, preferably one or two. One skilledin the art will appreciate the distinction between a monovalent alkenylgroup and a bivalent alkenyl group in view of the context of the term“alkenyl” in the definition for any particular substituent. When analkenyl is a terminal group, it will be monovalent, such as —CH═CH₂,—CH═CHCH₃, and the like. When an alkenyl is between other moieties, suchas “—C(O)—C₁₋₁₄alkenyl-C(O)—” in the definition of R¹, the alkenyl groupwill be bivalent, such as —CH═CH—, —CH═CHCH₂—, —CH₂CH═CHCH₂—, and thelike.

As used herein, the term “aryl” includes substituted or unsubstituted,monovalent or bivalent aromatic hydrocarbon groups. One skilled in theart will appreciate the distinction between a monovalent aryl group anda bivalent aryl group in view of the context of the term “aryl” in thedefinition for any particular substituent. When an aryl is a terminalgroup, it will be monovalent. When an aryl is between other moieties,such as “—C(O)—C₆₋₁₂aryl-C(O)—” in the definition of R¹, the aryl groupwill be bivalent.

Boc is t-butyloxycarbonyl.

Null with reference to a given substituent means that the substituent isabsent, and the chemical groups between which the substituent ispositioned are directly attached to each other by way of a covalentbond.

The compounds of Formulas (I), (II), (III), (IV) and (V) may have one ormore asymmetric carbon atoms, depending upon the substituents, and canhave stereoisomers, which are within the scope of the invention. Thecompounds of Formulas (I), (II), (III), (IV) and/or (V) can beadministered in the form of a pharmaceutically acceptable salt (e.g.,where M in the compounds of Formulas (I), (II), (III), (IV) and/or (V)is a pharmaceutically acceptable cation). The compounds of Formulas (I),(II), (III), (IV) and/or (V) can be administered in the form of apharmaceutically acceptable salt of a stereoisomer of the compounds.“Pharmaceutically acceptable salt” refers to salts which retain theirbiological effectiveness. Pharmaceutically acceptable base additionsalts can be prepared from inorganic and organic bases. Exemplarypharmaceutically acceptable salts derived from inorganic bases includesodium, potassium, lithium, ammonium, calcium and magnesium salts.Exemplary salts derived from organic bases include salts of primary,secondary and tertiary amines. Pharmaceutically acceptable acid additionsalts may be prepared from inorganic and organic acids. Exemplary saltsderived from inorganic acids include hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like.Exemplary salts derived from organic acids include acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, andthe like.

Methods for making the compounds of Formulas (I), (II), (III), (IV) and(V) are described in US Publication No. 2004/0006242, US Publication No.2003/0153532, US Publication No. 2002/0176861, US Publication No.2002/0049314, U.S. Pat. No. 6,551,600, U.S. Pat. No. 6,521,776, U.S.Pat. No. 6,290,973, and WO 03/099195, the disclosures of which areincorporated by reference herein in their entirety. Some compounds ofFormulas (I), (II), (III), (IV) and (V) and methods for making them arealso described by Hawkins et al, The Journal of Pharmacology andExperimental Therapeutics, 300(2):655-661 (2000); Lien et al, TheJournal of Biological Chemistry, 276(3):1873-1880 (2001); Przetak et al,Vaccine, 21:961-970 (2003); and Seydel et al, Eur. J. Immunol.,33:1586-1592 (2003), the disclosures of which are incorporated byreference herein in their entirety.

Exemplary compounds falling within the scope of the compounds ofFormulas (I)-(V) are set forth below.

No. Structure 112022

111230

111231

111232

111233

112043

112044

112047

112048

112049

112063

112064

112065

112066

112071

112072

112091

112092

112093

112098

112099

112100

112859

112860

112861

113634

113635

113643

113644

113651

113665

113666

118023

019772

118989

118999

119000

119001

118949

119327

119328

119329

119521

119522

119523

803028

803045

803056

803059

803592

803596

803597

803598

803599

803613

803731

803733

803751

803783

803784

803789

804061

804097

804121

804130

804221

804222

804252

804253

804281

804313

804339

804372

804503

804558

804596

804674

804678

804679

804732

804772

804947

804638

804666

804874

805028

805520 Isomer A

805270 Isomer B

805271

805274

805328

805329

805517

805518 Isomer A

805519 Isomer B

EXAMPLES Example 1 Intraperitoneal Administration of a TLR AgonistEnhances Therapeutic Efficacy of a Vaccine

To determine the effect of E6020 when administered intraperitoneallywith a cancer vaccine, e.g., granulocyte-macrophage colony stimulatingfactor (GM-CSF) secreting tumor cells, a mouse model using melanomacells was used. E6020 is a TLR-4 (Toll-like receptor-4) agonist. B6 mice(C57BL/6 mice) were engrafted subcutaneously with 1×10⁶ syngeneic B16F10murine melanoma cells. Three days after tumor cell inoculation, the micewere either (1) vaccinated subcutaneously (s.c.) with 1×10⁶ B16F10 tumorcells that were genetically modified to stably express and secretemurine GM-CSF (B16-GM-CSF cells); (2) vaccinated intraperitoneally(i.p.) with E6020; or (3) were treated with a combination of s.c. GM-CSFcell vaccination and i.p. E6020 (the GM-CSF cell vaccination and E6020vaccination were administered at separate sites in the mice). In theseexperiments, the GM-CSF cells were inactivated by gamma-irradiationprior to inoculation. Survival of the animals was monitored.

These experiments demonstrated that vaccination of the animals with theB16 GM-CSF cells and i.p. E6020 enhanced the therapeutic efficacy of theGM-CSF cells (FIG. 1).

Example 2 Local Administration of a TLR Agonist Enhances the TherapeuticEfficacy of a Cancer Vaccine

The effect of E6020 on treatment of B6 mice that were engraftedsubcutaneously with 1×10⁶ syngeneic B16F10 murine melanoma cells wasexamined. When tumors became palpable, the mice were injectedintratumorally with GM-CSF cells alone, or in combination with E6020(about 3-10 μg). Survival of the animals was monitored.

It was found that the population of animals treated intratumorally witha combination of GM-CSF cells and E6020 had increased survival comparedto animals that were untreated or treated with GM-CSF cells alone (FIG.2).

Example 3 MUC-1/E6020 Vaccine Therapeutic Effects

To test the effects of a MUC-1 vaccine with E6020 adjuvant for treatinginflammatory bowel disease (IBD) and subsequent development of colonadenocarcinoma, an engineered mouse strain that lacks the IL-10 gene andexpresses transgenic human MUC1 was used. Such mice spontaneouslydevelop intestinal inflammation resembling IBD followed by colonadenocarcinoma. These data were presented and published in Beatty etal., AACR Annual Meeting 2006, Washington D.C., Apr. 4, 2006.

In these experiments, mice were immunized intranasally with 30 mg/nareof Tn MUC100mer (HGVTSAPDTRPAPGSTAPPA)×5, SEQ ID NO:1) and 3 mg ofE6020. Animals were vaccinated at about 4.5 weeks and boosted at about6.5 weeks and 9 weeks.

MUC1 IL-10−/− mice treated with vaccine and E6020 had delayed onset ofIBD or did not develop IBD during the period of the experiment (FIG. 3).Mice treated with vaccine and E6020 had improved survival and did notdevelop colon cancer (FIG. 4 and Table 1).

TABLE 1 Treatment/no. of mice treated Age (weeks) Colon tumors Vaccine/6mice 14-35.5 0/6 Adjuvant/3 mice 12.5-18    3/3 No treatment/4 mice 8-15.5 4/4

The data demonstrate that the addition of E6020 to the MUC-1 vaccine canslow the progression to rectal prolapse associated with IBD, andsuppresses the appearance of histologically detected tumors.

Example 4 EGFRvIII Therapeutic Effects with Adjuvant

To determine whether an adjuvant can enhance the effect of an oncologyantigen (i.e., an antigen that can be used to vaccinate an animalagainst a cancer), C57/BL6J mice were immunized subcutaneously with atumor-associated peptide, LEEKKGNYVVTDHC (SEQ ID NO: 2) (derived from amutant form of EGFR, EGFRvIII) conjugated to the protein carrier keyholelimpet hemocyanin (KLH), with or without E6020 or murine GM-CSF, acytokine used in cancer vaccine trials to boost immune response. E6020was dosed at 3 μg, GM-CSF at 5 μg and the peptide-KLH conjugate at 25 μgper dose. Mice were immunized three times at intervals of three weeks.Serum was prepared from mice two weeks after each immunization andtested for EGFRvIII peptide-specific antibodies using ELISA on platescoated with EGFRvIII peptide conjugated to bovine serum albumin. Theresults in Table 2 are presented as titers from individual animals. Thetiter is defined as the last serum dilution at which a signal at 0.25 ODunits above background was observed.

The data from these experiments demonstrate that E6020 enhanced the meantiter of antigen-specific IgG2a, which binds high affinity Fc receptorsinvolved in antibody-dependent cell-mediated cytotoxicity (ADCC). IgG2ais the mouse correlate of the human IgG1 isotype that is used incurrently marketed human anti-tumor monoclonal antibodies, because it ismost efficacious in tumor killing.

The combination of E6020 and GM-CSF in the vaccination with EGFRvIIIpeptide demonstrated a greater effect on IgG2a titers than eithermaterial alone. These data demonstrate the usefulness of combinations ofE6020 with other immunoenhancers.

TABLE 2 Antibody titers to EGFRvIII peptide are enhanced by E6020Antigen administered with: PBS E6020 GM-CSF E6020/GMCSF Antibodysubclass: IgG1 IgG2a IgG1 IgG2a IgG1 IgG2a IgG1 IgG2a 6000 3000 30001500 12000 750 12000 3000 12000 3000 24000 6000 12000 750 12000 600024000 6000 24000 6000 12000 3000 24000 24000 24000 6000 24000 1200024000 3000 24000 24000 24000 6000 24000 12000 24000 6000 48000 2400024000 6000 24000 12000 48000 6000 48000 48000 48000 6000 24000 2400048000 6000 48000 48000 48000 24000 48000 48000 48000 24000 48000 48000Geometric mean 22008 6000 20182 10091 24000 3568 28541 20182 titer:

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

1. A method for stimulating an immune response in a subject individual,the method comprising the steps of: administering to the individual atleast one immunotherapeutic agent selected from one or more cancerantigens; one or more viral derived antigen associated with cancer; oneor more anti-cancer antibodies; and one or more anti-idiotypicantibodies to an anti-cancer antibody; and administering to theindividual one or more compound selected from formulae (I), (II), (III),(IV) and (V) and/or a pharmaceutically acceptable salt, hydrate,solvate, stereoisomer, amorphous solid thereof, or any combinationthereof.
 2. The method of claim 1, wherein the immunotherapeutic agentand the compound selected from formulae (I), (II), (III), (IV) and (V)are administered at about the same time.
 3. The method of claim 1,wherein the immunotherapeutic agent and the compound selected fromformulae (I) (II), (III), (IV) and (V) are administered separately. 4.The method of claim 1, wherein the immunotherapeutic agent and thecompound selected from formulae (I), (II), (III), (IV) and (V) areadministered to a subject individual at risk for developing cancer,diagnosed with a cancer, in treatment for cancer, or in post-therapyrecovery from cancer.
 5. The method of claim 4, wherein theimmunotherapeutic agent and the compound selected from formulae (I),(II), (III), (IV) and (V) are administered therapeutically incombination with a surgical procedure to remove or reduce the size of acancer tumor, radiation therapy, chemotherapy, and/or ablation therapy.6. The method of claim 4, wherein the immunotherapeutic agent and thecompound selected from formulae (I), (II), (III), (IV) and (V) areadministered therapeutically to stabilize a tumor by preventing orslowing the growth of the existing cancer, to prevent the spread of atumor or of metastases, to reduce the tumor size, to prevent therecurrence of treated cancer, or to eliminate cancer cells not killed byearlier treatments.
 7. The method of claim 3, wherein theimmunotherapeutic agent and the compound selected from formulae (I), II,(III), (IV) and (V) are administered in combination with cancertherapies.
 8. The method of claim 7, wherein the cancer therapy isdendritic cell therapy, chemokines, cytokines, tumor necrosis factors,TNF-α, chemotherapeutic agents, adenosine analogs, cladribine,pentostatin, alkyl sulfanates, busulfan, anti-tumoral antibiotics,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, mitomycin, aziridines, thiotepa, camptothecinanalogs, irinotecan, topotecan, cryptophycins, cryptophycin 52,cryptophicin 1, dolastatins, dolastatin 10, dolastatin 15, enedyineanticancer drugs, esperamicin, calicheamicin, dynemicin,neocarzinostatin, neocarzinostatin chromophore, kedarcidin, kedarcidinchromophore, C-1027 chromophore, epipodophyllotoxins, etoposide,teniposide, folate analogs, methotrexate, maytansinoids, maytansinol,maytansinol analogues, microtubule agents, docetaxel, paclitaxel,vinblastine, vincristine, vinorelbine, nitrogen mustards, chlorambucil,cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan,nitrosoureas, carmustine, lamustine, streptoxacin, nonclassicalkylators, altretamine, dacarbazine, procarbazine, temozolamide,platinum complexes, carboplatin, cisplatin, purine analogs, fludarabine,mercaptopurine, thioguanine, pyrimidine analogs, capecitabine,cytarabine, depocyt, floxuridine, fluorouracil, gemcitabine, substitutedureas, hydroxyurea, anti-angiogenic agents, canstatin, troponin I,biologic agents, ZD 1839, virulizin, interferon, antibodies andfragments thereof, anti EGFR, anti-HER-2/neu, anti-KDR, IMC-C225,anti-emetics, lorazepam, metoclopramide, domperidone, epithelial growthfactor inhibitors, transforming growth factor beta 1, anti-mucositicagents, dyclonine, lignocaine, azelastine, glutamine, corticoidsteroids, allopurinol, anti-osteoclastic agents, bisphosphonates,etidronate, pamidronate, ibandronate, osteoprotegerin, hormoneregulating agents, anti-androgens, LHRH agonists, anastrozole,tamoxifen, hematopoietic growth factors, anti-toxicity agents,amifostine and mixtures of two or more thereof.
 9. The method of claim1, wherein the immunotherapeutic agent and the compound selected fromformulae (I), (II), (III), (IV) and (V) are administeredprophylactically to the subject individual to prevent or delay thedevelopment of cancer.
 10. The method of claim 1, further comprisingadministering an immunostimulatory compound.
 11. The method of claim 10,wherein said immunostimulatory compound is a toll like receptor (TLR)agonist, TLR4, TLR7, TLR9, N-acetylmuramyl-L-alanine-D-isoglutamine(MDP), lipopolysaccharides (LPS), genetically modified and/or degradedLPS, alum, glucan, colony stimulating factors, EPO, GM-CSF, G-CSF,M-CSF, pegylated G-CSF, SCF, IL-3, IL6, PIXY 321, interferons,γ-interferon, α-interferon, interleukins, IL-2, IL-7, IL-12, IL-15,IL-18, MHC Class II binding peptides, saponins, QS2I, unmethylated CpGsequences, I-methyl tryptophan, arginase inhibitors, cyclophosphamide,or antibodies that block immunosuppressive functions, anti-CTLA4antibodies, or mixtures of two or more thereof.